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Essentials of Glycobiology Lecture 23 May 20th. 2004 Ajit Varki

Essentials of Glycobiology Lecture 23 May 20th. 2004 Ajit Varki. The “I-type” Lectins and the Siglecs. Current Classification of Lectins. Families with known protein sequence homologies Calnexin group (e.g., Calnexin, Calcireticulin, Calmegin)

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Essentials of Glycobiology Lecture 23 May 20th. 2004 Ajit Varki

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  1. Essentials of Glycobiology Lecture 23May 20th. 2004Ajit Varki The “I-type” Lectins and the Siglecs

  2. Current Classification of Lectins Families with known protein sequence homologies Calnexin group (e.g., Calnexin, Calcireticulin, Calmegin) *”L-type” lectins (e.g., (ERGIC-53 and VIP-36 in ER-Golgi pathway, Plant Lectins *"P-type" lectins (Mannose-6-Phosphate Receptors) *"C-type" lectins (e.g., Selectins, Collectins etc.) * Galectins(formerly "S-type" lectins) *"I-type" lectins (includes Siglec family) *”R-type" lectins (e.g., GalNAc-SO4 receptors, Plant Lectins) “Eglectins” (Frog Egg lectins) Eel Agglutinins (Fucolectins) Hyaluronan-binding proteins Ficolins Pentraxins Sequence homologies not known (Examples) CD11b/CD18 (beta3-integrin, CR3) Complement Factor H TNF, Interleukins & Cytokines Ameoba lectin Tachylectins Annexins Amphoterin *Have defined Carbohydrate Recognition Domains (CRDs)

  3. “I-type" Lectins Proteins other than antibodies that can mediate carbohydrate (glycan) recognition via immunoglobulin (Ig)-like domains Siglecs Sialic Acid Recognizing Ig-superfamily Lectins

  4. Proteins with Immunoglobulin(Ig)-like domains - phylogenetically similar to domains of vertebrate antibodies Occur in bacteria, but greatly expanded/diversified in animals. Ig-like domains traditionally classified into: V-set, related to Variable region of Antibodies C1- and C2-set, related to Constant region of Antibodies. Many cell adhesion molecules belong to Ig superfamily Until 1990s, Ig superfamily thought to mediate Protein:Protein interactions, but never Protein:Glycan interactions The Immunoglobulin Superfamily

  5. S S S S 1 V 1 V SS SS C2 2 C2 2 SS SS C2 3 3 C2 ss Hinge region Human IgG Heavy Chain SS SS Fc region SS SS Binding to Cells abolished by Sialidase Treatment! Tyrosine phosphorylation Association with SHP-1 CD22 - a B Cell specific cell surface molecule S S N 1 V Ig Superfamily member Found Primarily on mature resting sIgD Positive B Cells SS 2 C2 SS 3 C2 SS 4 C2 Gene disruption gives Altered B cell responses to antigens SS 5 C2 SS 6 C2 CD22-Fc : A TOOL FOR STUDYING CD22 FUNCTION SS 7 C2 C

  6. = Sialic acid GLYCOSPHINGOLIPID SIALIC ACIDS AT THE OUTER END OF SUGAR CHAINS ATTACHED TO VERTEBRATE GLYCOCONJUGATES S N-LINKED CHAIN O N Ser/Thr Asn O-LINKED CHAIN Secreted Protein S O N Membrane Protein Ser/Thr Asn OUTSIDE CELL MEMBRANE INSIDE

  7. SURFACE- OR METABOLICALLY- LABELLED GLYCOPROTEINS Sia Sia Sia DETERGENT LYSATE Sia Sia Sia SIALIDASE MILD PERIODATE TARGET CELL LINE PROTEIN A SEPHAROSE PNGaseF N-GLYCANS SIALIDASE MILD PERIODATE STRUCTURAL ANALYSIS SDS-PAGE CD22 RECOGNIZES N-GLYCANS ON A SUBSET OF SIALOGLYCOPROTEINS OF T- AND B-CELLS X

  8. Reproducible upon re-chromatography Destroyed by sialidase Requires alinked sialic acids Affected by the number and location of a-linked Sias Unaffected by sialic acids in other linkages e.g., a3-linked Destroyed by truncation of sialic acid side chains Does not require the tri-mannosyl core of N-glycans Identical results with natural and semi-synthetic glycans Kd for monovalent association of SiaaGalb-4Glc = m Kd for divalent association of SiaGal-4Glc = 1 m RECOGNITION OF SIALYLATED GLYCANS BY CD22

  9. Originally discovered by Crocker and Gordon as a lectin-like activity expressed on specific macrophage subpopulations and capable of binding to red blood cells Binding abolished by treatment of red cells with sialidase. Purified Sn from macrophages had Mr of 185kDa - adhered in sialic acid dependent manner to various lymphohematopoietic cells. Found concentrated in vivo at sites of interaction between macrophages and blood cells Purified Sn shown to directly recognize certain glycolipids and glycoproteins in a sialic acid-dependent manner Sialoadhesin (Sn)

  10. Human Siglecs Sialic acid-recognizing Ig-superfamily lectins) V-set domain C2-set domain Trp2 Neu5Ac Trp106 Arg97 3 CD33 Myeloid Precursors Monocytes Macrophages 4 MAG Glia G A F 10x 1 Sialoadhesin Macrophage Subset 2 CD22 B cells ?Basophils

  11. + + Sia2-6Gal1-4GlcNAc1- + + Sia2-3Gal1-(3)4GlcNAc1- Sia2-3Gal1-(3)4GlcNAc1- (4)3 1 Fuc + + + + Sia2-3Gal1-3GalNAc1- + Sia2-6GalNAc1- Biosynthetic pathways for sialylated chains recognized by mammalian lectins RECOGNIZED BY: GlcNAc1- CD22 Sn MAG CD33 Selectins Gal1-(3)4GlcNAc1- Gal1-3GalNAc1-R GalNAc1-R Siglec subfamily

  12. Myelin-Associated Glycoprotein (MAG) / Siglec-4 3 CD33 Myeloid Precursors Monocytes Macrophages 4 MAG Glia • Most highly conserved Siglec, from fish to man. Rat and human MAG sequences 96% identical, with only two differences in first 183 amino acids • Mediates cell-cell interactions between myelinating glial cells and neurons in CNS and PNS • Implicated in formation and maintenance of myelin • V-set domain recognizes a2-3-linked Sias on O-glycans or glycolipids - with extended binding site • MAG null mutants have defects in initiation of CNS myelination, formation of intact myelin sheaths and to a minor extent, integrity of myelin. In the PNS, only maintenance of myelin is impaired. • Glycosyltransferase KO eliminating glycolipid ligands gives similar phenotype. 10x 1 Sialoadhesin Macrophage Subset 2 CD22 B cells ?Basophils

  13. Human Siglecs (Sialic acid-binding Ig-superfamily lectins) Human Siglecs Sialic acid-recognizing Ig-superfamily lectins) ITIM ITAM Putative Tyr-based motif V-set domain C2-set domain CD33(Siglec-3)-related Siglecs 9 Monocytes Granulocytes T cell subset 6 OBBP-1 Placenta B-cells 7 AIRM-1 NK cells Monocytes T cell subset 8 SAF-2 Eosinophils 10 Monocytes Eosinophils B-cells 11 Macro- Phage Subset 5 Neutrophils Monocytes 3 CD33 Myeloid Precursors Monocytes Macrophages 4 MAG Glia 10x 1 Sialoadhesin Macrophage Subset 2 CD22 B cells ?Basophils

  14. Alignment of C-terminal Cytoplasmic Tails of CD33-related Siglecs reveals Two Conserved Tyrosine-containing Motifs • Adapted from: Crocker P. and Varki, A. • Trends in Immunology22: 337, 2001 Distal motif Proximal motif Classical ITIM Similar to (Thr-Ile-Tyr-X-X-(Val/Ile) in SLAM (signalling lymphocyte activation molecule) and SLAM-related proteins that bind SAP (SLAM-associated protein), a T-cell-specific molecule that inhibits SHP-2 recruitment to SLAM Immunoreceptor Tyrosine-based Inhibition Motif (Ile/Val/Leu/Ser)-X-Tyr-X-X-(Leu/Val) Phosphorylation by src-family kinases Recruitment of tyrosine phosphatases SHP-1 and SHP-2, sometimes also inositol phosphatase SHIP

  15. Myeloid progenitor Lymphoid progenitor NK cell CD16++ CD56– NK cell CD16+ CD56+ CD8+ T-cell subset Monocyte Baso- phil Eosin- ophil Neutro- phil B-cell Siglec-7 Siglec-9 Siglec-9 Siglec-10 CD33 Siglec-5 Siglec-7 Siglec-9 Siglec-10 (low) Siglec-5 Siglec-9 Siglec-7 Siglec-9 CD22? Siglec-8 (low) Siglec-8 Siglec-10 (low) Siglec-2 (CD22) Siglec-5 Siglec-6 Siglec-9 Siglec-10 Dendritic cell Macrophage Siglec-1 (Sialoadhesin) CD33 Siglec-5, Siglec-11 CD33 Siglec-7 Distribution of Human Siglecs in the Hematopoietic System(based on incomplete data available to date) Stem cell • Adapted from: Crocker P. and Varki, A. • Trends in Immunology22: 337, 2001 Siglec-3 (CD33)

  16. SIDE CHAIN (REQUIRED FOR BINDING OF MOST) NEGATIVELY CHARGED CARBOXYLATE (REQUIRED) CLEAVAGE SITE FOR SIALIDASE (NEURAMINIDASE) LINKAGE TO UNDERLYING SUGAR CHAIN (RELATIVE OR ABSOLUTE PREFERENCE) 5--N-ACYL-GROUP (AFFECTS BINDING OF SOME) Carbon Oxygen Nitrogen Hydrogen Structural Features of Sialic Acids Involved in Recognition by Siglecs - 9 7 1 8 6 2 5 3 4

  17. NH 2 Ig V-set domain (Sialic Acid Binding site) What exactly is the Mechanistic Connection? ITIM Motif (Tyrosine kinase Target site) Tyrosine Phosphatase Binding site (S/I/L/VxYxxL/V) Tyrosine kinase target?SAP Binding Site? (TxYxxI/V) A Generic Siglec Ig C2-set domain(s): 1 to 17 TM COOH

  18. P-B P-B: (Sialyl-LacNAc)n-PAA-Biotin P-B Mild periodate oxidation Sialidase Most Siglecs on Cell Surfaces are “masked” but can be uncovered by removing or altering cell surface sialic acids “Unmasking” can also occur spontaneously during cellular activation,via as yet unknown mechanisms

  19. Three Possible Models for Functions of CD22-Sialic Acid Interactions (Collins et al Glycobiology. 2002 12:563-71)

  20. iii iv Virally infected cell (eg influenza) Ligand expressing cell Ligand expressing cell Ligand expressing cell ? ? ? Siglecs Siglec expressing cell Siglec expressing cell Siglec expressing cell Sialic-acid- containing glycan Siglec expressing cell Desialylated glycan Constitutively Unmasked Masked Unmasked on activation or exposure to sialidase (e.g., viral infection) Unmasked on activation or exposure to Sialidase (e.g., viral infection) Reduced Adhesion/Signalling? CD33-related Siglecs? Cell-cell interactions (e.g., sialoadhesin) CD33-related Siglecs? Cell signalling (e.g., CD22) CD33-related Siglecs? Adhesion/Signalling? (e.g., CD22) CD33-related Siglecs? • Adapted from: Crocker P. and Varki, A. • Trends in Immunology22: 337, 2001 How Interactions of Siglecs with other cells might be affected by cis-interactions with sialylated glycoconjugates ii i

  21. Structural/Physical Roles Influenza Malaria Cholera Helicobacter Mycoplasma Rotavirus Polyoma virus Coronavirus Pertussis Tetanus etc. Ligands for Extrinsic Receptors EXTRINSIC RECEPTOR Molecular Mimicry SELF E.Coli Gonococcus Meningococcus Campylobacter Trypanosoma Streptococcus Etc. M ? INTRINSIC RECEPTOR M = Micro-organism/Toxin SIALYLATED GLYCAN = Biological Roles of Sialic Acids Siglecs Factor H Selectins Uterine Agglutinin Laminins Ligands for Intrinsic Receptors SELF

  22. Siglecs unmasking Sialic-acid- containing glycan Sialoadhesin expressing macrophage Siglec expressing cell Siglec expressing cell Desialylated glycan Increased pathogen uptake? Decreased pathogen survival? Decreased immune cell activation? Increased pathogen survival? Possible interactions of Siglecs with Sialylated pathogens • Adapted from: • Crocker P. and Varki, A. • Immunology103: 137-145,2001 Sialylated pathogen

  23. <1.0% “Great Apes” Neu5Ac Neu5Gc ~0.5% Pongo pygmaeus Gorilla gorilla Homo sapiens Pan paniscus Pan troglodytes Orangutan Gorilla Chimpanzee Human Bonobo Genetic Mutation Causing loss Of Neu5Gc MEAN Amino Acid Difference Evolutionary Relationships amongst Humans and the Great Apes 0 5 Millions of Years Before Present* 10 *Precise Timing Uncertain

  24. Alignment of Siglec Gene Clusters from 5 Species Kallikrein-Like (KLK) Genes are Highly Conserved but Siglec Genes are not Rodent Siglec Clusters have fewer Genes and Pseudogenes and are generally more conserved

  25. Human-Specific Features in the Siglec Gene Cluster Deletion of Siglec-13 Largest Number of Pseudogenes “Essential Arginine” Mutation in Siglec-XII

  26. Probable Orthologous Correspondences of CD33-related Siglecs Based on published and on-line data as of Dec 2003 Criteria for ortholog assignment include sequence similarity of amino-terminal V-set domains, map location, gene structure, and phylogenetic relationships. NF: Corresponding V-Set domain not found in available data on this species. * Siglec-like molecules missing Arg residue required for optimal Sia binding. # Located outside CD33-related Siglecs gene cluster. ? Published genomic information not sufficient to definitively determine status

  27. ITAM ITIM putative tyrosine-based motif Ig V-set Ig C2-set FNIII Transmembrane Siglecs Other I-type lectins Sn 1 L1 CD22 NCAM Siglec-3-related subfamily 2 ICAM-1 MAG Hemolin S2V 4 10 11 AIRM1 5 L1 OB-BP1 CD2 CD33 6 7 8 9 CD83 P0 3 ?

  28. Tissue distribution and glycan recognition of Non-Siglec I-type lectins CRD not defined in any of these

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